The ionization of large biomolecules by matrix assisted laser desorption in high vacuum (Matrix Assisted Laser Desorption and Ionization=MALDI for short) for mass spectrometric analyses has been known since the end of the 80s. This type of analyte molecule desorption also works in gases at atmospheric pressure. DE 196 08 963 (Franzen 1995) describes how analyte molecules at atmospheric pressure can be favorably desorbed by the use of special matrix substances in order to be then introduced to a subsequent ionization with protonating ions. This produces an extremely high yield of analyte ions. The subsequent ionization is carried out using protonating ions generated by electron beams, for example beams of beta particles, corona discharges or UV lamps. The analyte ions are predominantly singly charged, as is the case with the original vacuum MALDI. As already happens with the well-known method of electrospray ionization, the ions generated are transferred into the vacuum of the mass spectrometer through a capillary.
In the two practically identical inventions U.S. Pat. No. 5,965,884 (V. V. Laiko and A. L. Burlingame) and EP 0 964 427 A2 (J. Bai, S. M. Fischer, and J. M. Flanagan) the MALDI method including the ionization of the analyte molecules as such, which is well-known from high vacuum, is carried out at atmospheric pressure. As is the case with vacuum MALDI, the analyte ions here are also predominantly singly charged.
The disclosure WO 02/097 857 A1 (C. M. Whitehouse) also suggests the generation of analyte ions by MALDI at atmospheric pressure but within a high-frequency ion guide system.
The current way of elucidating the sequence of biopolymers such as proteins is to subject the chain-shaped analyte ions to a suitable fragmentation process and to then use the daughter ion spectra thus generated to read the sequence of the amino acids of the chain. For this process it is advantageous if the chains are fragmented between each of the amino acids. Surprisingly, it has proven difficult to evenly fragment the singly charged ions generated during the MALDI process. This is especially true of MALDI ions generated at atmospheric pressure, which are immediately cooled in the ambient gas and hence have no excess intrinsic energy to bring into the fragmentation process. Soft fragmentation, such as occurs during multiple collision processes in ion traps, and also in multiple photon processes (IRMPD—Infrared Multiphoton Dissociation), is extremely unsuccessful here.
In contrast, doubly or higher charged analyte ions, such as those preferredly generated by electrospray ionization, are much easier to fragment between each amino acid even though they are also generated at atmospheric pressure. Collisional ion dissociation (CID) provides daughter ion spectra which can be used quite well to interpret the sequence. Daughter ion spectra fragmented from doubly charged biomolecules by electron capture dissociation (ECD) are even better for elucidating the structure and determining the sequence since, in this case, the daughter ion spectra generated are particularly easy to interpret.
For multiply charged negative ions, the EDD process can be used in a similar way to generate informative daughter ion spectra (EDD=electron detachment dissociation). This shoots an electron out of multiply charged negative ions and also leads to evenly distributed fragmentation.